Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells
Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells
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DOI：
10.1261/rna.224606
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发表时间：
2006-12-01
期刊：
影响因子：
4.5
通讯作者：
Sharp, Phillip A.
中科院分区：
文献类型：
--
作者：
Calabrese, J. Mauro;Sharp, Phillip A.
哺乳动物RNA干扰（RNAi）的研究主要集中在microRNA的作用;然而，在其他生物体中，内源性短干扰RNA（siRNA）参与沉默过程。迄今为止，在哺乳动物细胞中难以表征类似的分子。P19是RNA沉默的植物抑制子，以高亲和力结合siRNA。在这里，在小鼠胚胎干细胞（ES）中P19结合的短RNA的特点。我们发现，P19选择性免疫沉淀内源性短RNA从ES细胞。免疫沉淀RNA的克隆揭示了对与核糖体RNA（rRNA）精确匹配的短RNA的强选择，其中特定的短rRNA种类高度富集于P19免疫沉淀物中。富集的rRNA的互补链没有被克隆，这令人惊讶，因为之前认为P19仅结合SiRNA。我们发现，P19紧密结合到一个非典型的dsRNA底物组成的短RNA退火到一个更长的合作伙伴，这样的双链区域之间的两个是19个碱基对长。结合到类似的内源性物种可能解释协会的P19与短rRNA在ES细胞。最后，我们发现富含P19的rRNA不参与典型的RNAi，因为它们在没有Dicer的情况下存在，并且不起转录后基因沉默剂的作用。我们的结果支持了先前的观察，即内源性siRNA在小鼠ES细胞中不是丰富的分子。
Studies of mammalian RNA interference (RNAi) have focused largely on the actions of microRNAs; however, in other organisms, endogenous short-interfering RNAs (siRNAs) are involved in silencing processes. To date, similar molecules have been difficult to characterize in mammalian cells. P19 is a plant suppressor of RNA silencing that binds with high affinity to siRNAs. Here, the short RNAs bound by P19 in mouse embryonic stem (ES) cells have been characterized. We show that P19 selectively immunoprecipitates endogenous short RNAs from ES cells. Cloning of immunoprecipitated RNA reveals a strong selection for short RNAs that are exact matches to ribosomal RNA (rRNA), with particular short rRNA species highly enriched in P19 immunoprecipitates. Complementary strands to the enriched rRNAs were not cloned, which was surprising because P19 was previously thought to bind only siRNAs. We show that P19 binds tightly to a noncanonical dsRNA substrate comprised of a short RNA annealed to a much longer partner, such that the double-stranded region between the two is 19 base pairs long. Binding to similar endogenous species might explain the association of P19 with short rRNAs in ES cells. Finally, we show that the P19-enriched rRNAs are not involved in canonical RNAi, as they exist in the absence of Dicer and do not function as post-transcriptional gene silencers. Our results support the previous observation that endogenous siRNAs are not abundant molecules in mouse ES cells.